Method and apparatus to coat a substrate uniformly with a small volume of fluid

ABSTRACT

A method is provided for applying a fluid onto a surface of a solid substrate. The method employs a distribution member having an upper surface and a lower surface, and including a permeable portion having channels therethough. The distribution member is positioned such that the lower surface of the distribution member is in opposing relation to the surface of the substrate. Once the distribution member is in position, the fluid is dispensed onto the upper surface of the permeable portion of the distribution member such that the fluid penetrates the distribution member and is retained thereby. In addition, a distribution pressure differential is applied between the upper and lower surfaces without using mechanical means such as a squeegee so that a portion of the fluid passes through the channels of the permeable portion of the distribution member and onto the solid substrate surface. Other embodiments of the invention include an apparatus for carrying out the aforementioned method and a reagent application station for applying a plurality of reagents onto a surface of a solid substrate.

TECHNICAL FIELD

[0001] This invention relates generally to a method and apparatus tocoat a substrate with a small volume of fluid. More particularly, theinvention relates to a method and apparatus to quickly and uniformlycoat a fluid such as reagents or fluids used in DNA array fabricationonto a surface of a solid substrate by providing a pressure differentialbetween substantially parallel surface of a fluid-containingdistribution member.

BACKGROUND

[0002] Nucleic acid hybridization is a known method for identifyingspecific sequences of nucleic acids; hybridization involves base-pairingbetween complementary nucleic acid strands. When single-stranded nucleicacids are used as probes to identify specific target sequences ofnucleic acids, probes of known sequences are exposed to and incubated insample solutions containing sequences to be identified. If a sequencehybridizes to a probe of a known sequence, the sequence is necessarilythe specific target sequence. Various aspects of this method have beenstudied in detail. In essence, all variations allow complementary basesequences to pair and thus form double-stranded stable molecules, and avariety of methods are known in the art to determine whether pairing hasoccurred, such as those described in U.S. Pat. No. 5,622,822 to Ekeze etal. and U.S. Pat. No. 5,256,535 to Ylikoski et al.

[0003] Hybridization of surface-bound probes to solution-based targetsis an effective means to analyze a large number of DNA or RNA moleculesin parallel. Specific probes of known sequences are attached to thesurface of a solid substrate in known locations. The probes are usuallyimmobilized on a solid support having a surface area of typically lessthan a few square centimeters. The solid support is typically a glass orfused silica slide which has been treated to facilitate attachment ofprobes. A mobile-phase sample containing labeled targets, e.g., abuffered aqueous solution containing target DNA, is contacted with andallowed to react with the surface. By detecting the labels to determinewhether hybridization has occurred at specific locations, it is possibleto determine the composition of the sample and the sequences of theunknown targets. Alternatively, target biomolecules may be bound to thesurface while labeled probes are contained in the mobile phase. Ineither case, the hybridization reaction typically takes place over atime period that can be many hours, for a typical sample containingtarget material in the concentration range in the picomolar domain.

[0004] In the preparation of arrays such as those for use in nucleicacid hybridization, reagents may be applied to predetermined locationson the surface of a substrate. Generally, a surface is first cleaned orotherwise prepared by exposure to a fluid containing a reagent. Then,array preparation will involve application of biomolecule-containingfluids at discrete locations. For nucleic-acid probe array preparation,the biomolecule-containing fluid may contain the already-formed probesthat can bind with the surface, or a specific nucleotide that will laterconstitute a portion of a probe that is synthesized in situ on thesurface. Then, treatment of a portion of or the entire surface with adifferent fluid may follow. The steps may be repeated a number of timesin situ to prepare the desired array. Once an array of probes is formedon a substrate surface for hybridization with target molecules in asample fluid, hybridization may be carried out by uniformly exposing theentire substrate surface to the sample fluid.

[0005] It is apparent, then, that surface coating by a fluid is animportant aspect in array technology, particularly in the field ofbiomolecular arrays. Important aspects of coating procedures include theamount of fluid used and the rate of throughput. In general, coatingprocedures should employ only a small quantity of fluid, for a number ofreasons. First, the fluid may contain expensive or rare reagents, andwaste of such fluids is undesirable. Second, many ordinary reagents thatare used in array preparation are toxic, and decreasing their use isdesirable in order to lower the risk of human exposure. A highthroughput rate also implies that it takes less time to coat eachsubstrate surface, thereby also lowering the risk of human exposureduring the coating procedure.

[0006] Another important aspect of coating procedures is uniformity ofcoverage. For biomolecular arrays, it is desirable to uniformly apply afluid onto a substrate surface to ensure that each feature is attachedor formed under similar conditions. In addition, during use of a formedarray containing surface-bound probes, uniform distribution of samplefluid to ensure proper hybridization is necessary. Without uniform fluiddistribution, resultant hybridization data will be compromised.

[0007] One method by which a surface may be coated with a small amountof fluid is through the use of a flow cell assembly. Variations on theuse of a flow cell are described in U.S. Pat. Nos. 4,596,695 toCottingham and 5,145,784 to Cox et al. The basic flow cell methodtypically provides that a cover and substrate are positioned parallel toeach other. A gap is thus formed between the cover and the substrate. Tocontrol the size of the gap, one or more spacers having a selectedheight are disposed within the gap. In addition, the cover, the spacersand the substrate are arranged such that a chamber is provided having aninlet channel and an outlet channel. By creating an appropriate pressuregradient between the inlet and outlet channels, fluid fills the chamberby laminar flow, coating the surface of the substrate within thechamber. By controlling the volume in the chamber through the properselection of the spacer height, the amount of fluid needed to coat thesurface can be reduced.

[0008] The use of the flow cell method has a number of drawbacks. First,uniform coating requires laminar flow of the fluid. Laminar flow regimegenerally implies that there is an absolute upper limit to thevolumetric rate given the geometry of the chamber. Second, to increasethe flow rate of the fluid, the pressure gradient between the inlet andoutlet channels must be increased. However, pressure surges that aregenerated while increasing the pressure gradient tend to cause the flowcell assembly to leak, either at the cover/support interface or at thesupport/substrate interface. Third, any irregularity in the surfaceprofile of the substrate tends to disrupt laminar flow. As a result, airpockets may be formed and trapped within the flow cell assembly thatwill interrupt contact between the fluid and the substrate. Thus, whilethe use of a flow cell tends to lower the amount of reagent fluid waste,the gain in lowered waste is offset by diminishing throughput.

[0009] Spin coating may be employed to quickly and uniformly coat afluid on a substrate. Spin coating is usually performed by dispensingthe fluid at or near the center of a substrate. The substrate is spuneither during or after the reagent is dispensed such that the fluidspreads radially and outwardly to cover the entire substrate. In thismethod, the volume needed to cover a surface depends on fluid property,e.g., viscosity and surface tension, and the surface energy of thesubstrate. When a low energy surface is provided, a relatively largevolume fluid is needed to cover the entire surface. Without sufficientvolume, applied fluid tends to exhibit clustering behavior and does notcover the entire surface uniformly. Thus, a relatively large amount offluid is first applied to the surface at a low spin speed to cover theentire surface. Then, the substrate is spun at a higher speed to removeexcess fluid. Consequently, while spin coating may be advantageous interms of high throughput, it is a relatively wasteful technique.

[0010] Thus, there is a need to provide a method and apparatus to coat asubstrate surface with a small volume of fluid quickly and uniformlywithout relying on spin coating or an ordinary flow cell.

SUMMARY OF THE INVENTION

[0011] Accordingly, it is an object of the present invention to overcomethe above-mentioned disadvantages of the prior art by providing a methodfor coating a substrate surface with a small volume of fluid withoutgenerating excess waste.

[0012] It is another object of the invention to provide such a methodwherein the substrate is coated quickly and uniformly.

[0013] It is still another object of the invention to provide anapparatus for use in carrying out the aforementioned method.

[0014] It is a further object of the invention to provide such anapparatus for use in carrying out the aforementioned method with aplurality of fluids in succession.

[0015] Additional objects, advantages and novel features of theinvention will be set forth in part in the description which follows,and in part will become apparent to those skilled in the art uponexamination of the following, or may be learned by practice of theinvention.

[0016] In one general aspect, then, the present invention relates to amethod for applying a fluid onto an application area located on asurface of a solid substrate. The method comprises providing adistribution member having an upper surface and a lower surface, andincluding a permeable portion having channelss of a selected size. Thedistribution member may be generally planar and is positioned such thatthe lower surface of the distribution member is in generally opposingspacing relation to the surface of the substrate. The distributionmember and the substrate may also be substantially uniformly spacedrelation. A predetermined volume of the fluid is dispensed onto theupper surface of the permeable portion of the distribution member suchthat the fluid penetrates and is retained by the permeable portion ofthe distribution member. The fluid may penetrate and be retained by thepermeable portion of the distribution member under the application of aloading pressure differential between the upper and lower surfaces. Inaddition, a distributing pressure differential is applied between theupper and lower surfaces generally without using a movable mechanicalmeans designed to spread fluids such as a squeegee such that at least aportion of the fluid passes through the channels of the permeableportion of the distribution member and onto the application area. Theapplication area may contain any array of biomolecules covalently orotherwise attached thereto.

[0017] In another aspect, the invention relates to the above methodwherein a perimeter spacer is provided and positioned between and incontact with the lower surface of the distribution member and thesurface of the solid substrate. Accordingly, the perimeter spacerdefines an application volume between the lower surface of thedistribution member and the surface. The fluid passes through thechannels of the permeable portion of the distribution member and intothe application volume.

[0018] In still another aspect, the invention relates to the abovemethod wherein the distribution pressure differential is generated byraising the pressure at the second surface of the distribution member.The distribution pressure differential may also be generated by loweringthe pressure at the lower surface of the distribution member.

[0019] In a further aspect, the invention relates to the above methodfurther comprising the step of dispensing a second fluid onto the uppersurface of at least the permeable portion of the distribution member. Apressure differential between the upper and lower surfaces is appliedsuch that the second fluid passes through the channels in the permeableportion of the distribution member and onto the application area,displacing the fluid away from the solid substrate surface. In addition,the method may further comprise the step of flushing a gas through thepermeable portion of the distribution member such that the second fluidis displaced away from the solid substrate surface. The gas may comprisenitrogen or argon.

[0020] In a still further aspect, the invention relates to the abovemethod wherein the fluid contains water, or an organic solvent such asacetonitrile, an alcohol, or a ketone. Likewise, the fluid may comprisea biomolecule. Examples of biomolecules include oligonucleotides,polynucleotides, oligopeptides and polypeptides. In order to preventfluid waste, it is preferred that the predetermined volume of the fluiddoes not substantially exceed the application volume. More preferably,the predetermined volume of the fluid should not exceed about 150% ofthe application volume. Still more preferably, the predetermined volumeshould not exceed about 110% of the application volume.

[0021] In another general aspect, the invention relates to an apparatusfor applying a fluid onto a surface of a solid substrate. The apparatuscomprises a distribution member having an upper surface, a lower surfaceand a permeable portion formed by a plurality of channels extending fromthe upper surface to the lower surface. Such a distribution member maycomprise a perforated flat piece or a mesh. Affixed in sealed contactwith the upper surface about the permeable portion of the distributionmember is an enclosing wall that, together with the distribution member,defines an enclosure having an enclosure volume. The apparatus alsoprovides means for positioning the distribution member in relation tothe solid substrate surface such that the lower surface of thedistribution member is in generally uniformly spaced opposingrelationship to the solid substrate. Once fluid is introduced onto thefluid distribution area within the enclosure, a means for producing apositive pressure differential between the upper surface and the lowersurface of the distribution member can be activated. As a result, theliquid passes through the member and onto the solid substrate surface.

[0022] In another aspect, the invention relates to the above apparatuswherein a spacer having generally parallel upper and lower surfaces isprovided. In such a case, the upper surface of the spacer is affixed tothe lower surface of the distribution member about the permeableportion. The lower surface is placed in contact with the substrate suchthat an application space having an application volume is substantiallyenclosed by the spacer, the lower surface of the distribution member,and the substrate surface. An opening may be disposed in the spacer suchthat the application space fluidly communicates with open air. In orderto prevent fluid waste, it is preferred that the enclosure volume doesnot substantially exceed the application volume. More preferably, theenclosure volume should not exceed about 150% of the application volume.Still more preferably, the enclosure volume should not exceed about 110%of the application volume.

[0023] In still another aspect, the invention relates to the aboveapparatus wherein the means for introducing the fluid comprises a fluidsource for supplying the fluid. A fluid transfer channel is connected tothe enclosure and, a fluid valve is disposed between the fluid sourceand the fluid transfer channel. Fluid communication is provided from thefluid source to the fluid transfer channel when the fluid valve is open.In addition, the apparatus may include means for introducing a secondfluid. Such means may comprise a second fluid source for supplying thesecond fluid and a second fluid valve disposed between the second fluidsource and the fluid transfer channel, where the second fluid source isin fluid communication with the fluid transfer channel when the secondfluid valve is open. In any case, the fluid may contain a liquid such aswater, acetonitrile, an alcohol, or a ketone for use in facilitatingchemical reactions. Where hybridization reactions are desired, the fluidwill contain a biomolecule such as an oligonucleotide, polynucleotide,oligopeptide, or polypeptide.

[0024] In a further aspect, the invention relates to the above apparatuswherein the mean for producing a pressure differential may comprisemeans for raising pressure within the chamber. Such pressure raisingmeans may comprise means for introducing a gas into the enclosure from apressured source. The gas may comprise, for example, nitrogen or argon.

[0025] In still another general aspect, the invention relates to aregent application station for applying a plurality of reagents onto asurface of a solid substrate. The apparatus comprises a distributionmember having an upper surface, a lower surface and a permeable portionformed by a plurality of channels extending from the upper surface tothe lower surface. An enclosure is formed by the upper surface of thedistribution member and an enclosing wall affixed about the permeableportion in sealed contact with the upper surface of the distributionmember. The apparatus also provides means for positioning thedistribution member in relation to the solid substrate surface such thatthe lower surface of the distribution member is in generally uniformlyspaced opposition relation to the solid substrate. To control pressurewith enclosure, a variable pressure pump is provided having an inlet foreach reagent and an outlet in fluid communication with the enclosure. Tosupply the reagents, a source for each reagent is provided, and a valveis disposed between each inlet and source to provide individual controlover fluid dispensing. It is preferred that no two valves are open atthe same time.

BRIEF DESCRIPTION OF THE FIGURES

[0026] The invention is described in detail below with reference to thefollowing drawings:

[0027]FIG. 1 schematically illustrates an apparatus of the presentinvention.

[0028]FIGS. 2A and 2B illustrate alternative embodiments of thedistribution member suitable for use in an apparatus of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0029] Before describing the invention in detail, it must be noted that,as used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a fluid”includes more than one fluid, reference to “a biomolecule” includes aplurality of biomolecules, reference to “a fluid source” includes aplurality of fluid sources and the like.

[0030] In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

[0031] The terms “array” is used herein to refer to an ordered patternof features, typically but not necessarily biomolecules, adherent to asubstrate, e.g., a plurality of molecular probes bound to a substratesurface and arranged in a spatially defined and physically addressablemanner. Such probes may be comprised of oligonucleotides, peptides,polypeptides, proteins, antibodies, or other molecules used to detectsample molecules in a sample fluid.

[0032] The term “biomolecule” as used herein refers an organic moleculethat may be found in a living organism or synthetically produced.Typically, biomolecules are large and may have a complementarycounterpart. Examples of biomolecules include but are not limited tonucleotidic molecules such as oligonucleotides and polynucleotides andpeptidic molecules such as oligopeptides and polypeptides.

[0033] The term “feature” refers to an element or a constituent part ofmatter forming a pattern situated on a surface. As used herein, featurescan be deposited, dispensed, printed, placed, positioned or otherwisedisposed on a surface.

[0034] The term “fluid” as used herein refers to a material that is notpurely gaseous which tends to flow to conform to the outline of itscontainer. Unless otherwise stated, the fluids described herein comprisea liquid and may contain solvated gas or fully solvated, partiallysolvated or suspended solids.

[0035] The term “hybridization” as used herein means binding betweencomplementary or partially complementary molecules, as between the senseand anti-sense strands of double-stranded DNA. Such binding is commonlynon-covalent in nature, and is specific enough that such binding may beused to differentiate between highly complementary molecules and othersless complementary. Examples of highly complementary molecules includecomplementary oligonucleotides, DNA, RNA, and the like, which comprise aregion of nucleotides arranged in the nucleotide sequence that isexactly complementary to a probe; examples of less complementaryoligonucleotides include ones with nucleotide sequences comprising oneor more nucleotides not in the sequence exactly complementary to a probeoligonucleotide.

[0036] The term “monomer” as used herein refers to a chemical entitythat can be covalently linked to one or more other such entities to forman oligomer. Examples of “monomers” include nucleotides, amino acids,saccharides, peptides, and the like. In general, the monomers used inconjunction with the present invention have first and second sites(e.g., C-termini and N-termini, or 5′ and 3′ sites) suitable for bindingto other like monomers by means of standard chemical reactions (e.g.,condensation, nucleophilic displacement of a leaving group, or thelike), and a diverse element which distinguishes a particular monomerfrom a different monomer of the same type (e.g., an amino acid surfacechain, a nucleotide base, etc.). The initial substrate-bound monomer isgenerally used as a building-block in a multi-step synthesis procedureto form a complete ligand, such as in the synthesis of oligonucleotides,oligopeptides, and the like.

[0037] The terms “nucleoside” and “nucleotide” are intended to includethose moieties which contain not only the known purine and pyrimidinebases, but also other heterocyclic bases that have been modified. Suchmodifications include methylated purines or pyrimidines, acylatedpurines or pyrimidines, or other heterocycles. In addition, the terms“nucleoside” and “nucleotide” include those moieties that contain notonly conventional ribose and deoxyribose sugars, but other sugars aswell. Modified nucleosides or nucleotides also include modifications onthe sugar moiety, e.g., wherein one or more of the hydroxyl groups arereplaced with halogen atoms or aliphatic groups, or are functionalizedas ethers, amines, or the like. Moreover, the terms “nucleoside” and“nucleotide” include functional analogs (whether synthetic or naturallyoccurring) of such sub units which in the polymer form (as apolynucleotide) can hybridize with naturally occurring polynucleotidesin a sequence specific manner analogous to that of tow naturallyoccurring polynucleotides. For example, these include the sub-units ofPNA and other polynucleotides as described in U.S. Pat. No. 5,948,902and references cited therein (all of which are incorporated herein byreference), regardless of the source.

[0038] The term “oligomer” is used herein to indicate a chemical entitythat contains a plurality of monomers. As used herein, the terms“oligomer” and “polymer” are used interchangeably, as it is generally,although not necessarily, smaller “polymers” that are prepared using thefunctionalized substrates of the invention, particularly in conjunctionwith combinatorial chemistry techniques. Examples of oligomers andpolymers include polydeoxyribonucleotides, polyribonucleotides, otherpolynucleotides which are—or C-glycosides of a purine or pyrimidinebase, polypeptides, polysaccharides, and other chemical entities thatcontain repeating units of like chemical structure.

[0039] The term “probe” as used herein means a biomolecule of knownidentity that is typically but not necessarily adherent to a substrateor a base member of a hybridization package.

[0040] The term “reagent” as used herein means a substance used in thepreparation of an array on a surface because of its chemical orbiological activity. Typically, reagents as used herein refer to liquidcompounds or solutions involved the synthesis of biomolecular arrays andinclude, but are not limited to: acetonitrile; buffered solutionscontaining biomolecules; mecaptosilane containing solutions; aqueousiodine solutions; alcohols; acids; bases; oxidizing agents; reducingagents; organic or inorganic fluids for deprotection reactions employedin in situ DNA synthesis; etc.

[0041] The term “sample” as used herein relates to a material or mixtureof materials, at least partially in fluid form, containing one or morecomponents of interest.

[0042] The term “squeegee” as used herein means an implement having anelastic surface that is used in printing for spreading, pushing, orwiping a liquid material on, across, or off a surface. Unless otherwisespecified, “squeegeeless means” as used herein refer to a means thatexcludes the substantial participation of a blade or other like solidcomponent for spreading, pushing, or wiping of a liquid material bydirect mechanical action.

[0043] The term “target” refers to a known or unknown molecule in asample, which will hybridize to a probe if the target molecule and themolecular probe contain complementary regions. In general, the targetmolecule is a “biopolymer,” i.e., an oligomer or polymer such as anoligonucleotide, a peptide, a polypeptide, a protein, an antibody, orthe like.

[0044] The present invention in general terms is directed to a methodfor applying a fluid onto an application area on a surface of a solidsubstrate. Unlike previous methods such as spin coating or flow celltechniques, the present method provides for fast and controlled deliveryof a fluid to coat a surface with little if any waste of the fluid. Themethod employs a generally planar distribution member to hold apredetermined volume of the fluid above the application area. Withoutthe use of a direct mechanical action by a solid component such as asqueegee, a pressure differential is applied between the upper and lowersurfaces of the member to effect flow of the fluid onto the applicationarea of the surface.

[0045] The invention is described herein with reference to the figures.The figures are not to scale, and in particular, certain dimensions maybe exaggerated for clarity of presentation. FIG. 1 schematicallyillustrates an apparatus of the present invention. As shown, theapparatus 10 comprises a distribution member 100 having an upper surface101 and a lower surface 109. The distribution member includes apermeable portion 105 with a plurality of channels 106 therethrough. Thechannels provide communication between the upper and lower surfaces ofthe distribution member. The apparatus also comprises an enclosure 200formed by a tapered cone-shaped wall 210 affixed about the permeableportion and in sealed relation to the upper surface of the distributionmember. As shown, the apparatus 10 further comprises a spacer 300 havinga generally parallel upper surface 301 and lower surface 309. The uppersurface of the spacer is affixed to the lower surface of thedistribution member about the permeable portion. The lower surface ofthe spacer is placed in contact with an upper surface 501 of a solidsubstrate 500. This is done by using means (not shown) for positioningthe distribution member in relation to the solid substrate surface suchthat the lower surface of the distribution member is in generallyuniformly spaced opposing relation to the solid substrate. Such meansare known to one of ordinary skill in the art and include, but are notlimited to, pulleys, levers, gears, and combinations thereof. Inparticular, indexing means typically used in semiconductor waferprocessing may be employed to control positioning with precision andaccuracy. Typically, a chuck of some type (not shown), e.g., vacuum,electrostatic, mechanical, etc., is used to render the solid substrateimmobile while the distribution member is positioned to ensure properalignment with the substrate surface. As a result, the lower surface ofthe distribution member, the spacer, and the substrate substantiallyenclose an application space 400. An optional hole 350 is provided inthe spacer such that an application area 505 located on the surfacewithin the application space fluidly communicates with open air. Theapplication area is the area on the surface of the substrate wheredesired reagents are applied in order to carry out desired reactions.Examples of such reactions include, but are not limited to,functionalization of the surface, in situ chemical synthesis such as ofbiomolecules in an array, and hybridization of surface bound probes witha sample fluid containing target biomolecules. It is envisioned that thefluid may be applied to the application area before, during, or afterthe formation of a particular biomolecular array and that the featuresof the array may be attached to the application area by covalentbonding, electrostatic bonding, polar attraction, Van Der Waal's forcesor other approaches known to one of ordinary skill in the art.

[0046] As shown, the enclosure formed by the wall 210 is generally inthe form of a cone. For such a conically shaped enclosure, the anglebetween the enclosing wall and the distribution member, i.e., the baseand the surface of the cone, can be any acute angle between 0° and 90°.It is expected that an angle of about 10 to about 30° is preferred dueto fluid flow, geometric and volume consideration. An angle of about 10to about 20° is more preferred, and about 15° should provide optimalresults. As shown, a transfer port 601 is disposed on the enclosingwall. Extending from the transfer port is a fluid transfer channel 600.The apparatus also provides for a source 610 of a fluid. A fluid valveis positioned between the fluid source and the fluid transfer channelsuch that when the fluid valve is open, the fluid source is in fluidcommunication with the fluid transfer channel, which in turn, fluidlycommunicates with the enclosure 200. Preferably, the fluid sourceimposes a loading pressure on the fluid such that when the fluid valveis open, the fluid flows from the source and fills the enclosure,thereby contacting the upper surface of the distribution member. Theloading pressure also effects penetration or retention of the fluid bythe distribution member. Once penetration and retention have beenachieved, the fluid valve is closed.

[0047] The apparatus also provides for a source of pressurized gas 630.Disposed between the pressurized gas source and the fluid transferchannel is a gas valve 631 that can regulate the pressure of the gasfrom the gas source. When the gas valve is open such that fluidcommunication is provided between the gas source and the enclosure, thepressurized gas is forced through the fluid transfer channel and intothe enclosure. As a result, pressure within the enclosure is raised withrespect to the pressure at the lower surface of the distribution member,and at least a portion of the fluid passes through the channelss of thepermeable portion of the distribution member and onto the applicationarea within the application volume. When the application volume 400 isfilled, the combination of the pressure from the pressurized gas and thephysical contact between the distribution member and the fluid ensuresthat all of the application area is substantially equivalently exposedto the fluid. No squeegee or other like means is employed or necessaryin this process to induce the pressure differential between the upperand lower sides of the distribution member. Once sufficient time haspassed, the gas valve may be opened further such that gas is flushedthrough the permeable portion of the distribution member and at least aportion of the fluid is thereby displaced away from the solid substratesurface.

[0048] Particularly in biomolecular array applications, it is arequirement that the gas used is pure, i.e., does not contain anycontaminants or impurities that could interfere with the function of thefluid. Particulate matter is particularly problematic because suchmatter may become lodged in the distribution member, thereby adverselyaffecting fluid flow. Suitable gases include, but are not limited toair, nitrogen, argon and other inert gases.

[0049] Where it is desired to apply a second fluid after the applicationof the initial fluid, a second fluid source 620 is provided as shown inFIG. 1. Disposed between the second fluid source and the fluid transferchannel is a second fluid valve 621. After the initial fluid isdisplaced away from the solid substrate surface, the second fluid valveis opened to render the second fluid source in fluid communication withthe fluid transfer channel. Preferably, the second fluid source, likethe fluid source, also imposes a loading pressure on the second fluidsuch that when the second fluid valve is open, the second fluid flowsfrom its source, fills the enclosure, and contacts the upper surface ofthe distribution member. The loading pressure also facilitatespenetration and retention of the second fluid by the distributionmember. Once penetration and retention have been achieved and the secondfluid valve is closed, gas is introduced into the enclosure to cause thesecond fluid to flow onto the application area within the applicationvolume. In the alternative, the second fluid may be a wash fluid that isflushed through the permeable portion of the distribution member todisplace the fluid from the surface of the solid substrate.

[0050] It is evident, then, that a method of applying one or more fluidsis provided. One step of the method involves applying a distributionpressure differential between the upper and lower surfaces of thedistribution member for each fluid. Such differential can be applied ina number of ways. For example, pressure at the upper surface of thedistribution member may be raised. In addition, pressure at the lowersurface of the distribution member may be lowered. Furthermore, acombination of pressure raising at the upper surface of the distributionmember and pressure lowering at the lower surface of the distributionmember may be employed. Means for controlling pressure usually involvethe use of a pump as is generally known in the art.

[0051] It is also evident that the invention encompasses a reagentapplication station for applying a plurality of reagent fluids onto asurface of a solid substrate. The station comprises: a distributionmember having an upper surface, a lower surface and a permeable portionformed by a plurality of channels extending from the upper surface tothe lower surface; an enclosure formed by an enclosing wall affixedabout the permeable portion in sealed contact with the upper surface ofthe distribution member; and means for positioning the distributionmember in relation to the solid substrate surface such that the lowersurface of the distribution member is in generally uniformly spacedopposition relation to the solid substrate. The station also comprises asource for each reagent; a variable pressure pump having an inlet foreach source and an outlet in fluid communication with the enclosure; anda valve between each inlet and each source. The variable pressure pumpshould be capable of generating at least two pressures within theenclosure to effect loading of fluid into the distribution member anddispensing of fluid onto the application area of the substrate surfaceas described above. Such a pump may be able to provide a continuousrange of pressures within the enclosure. It is also preferred that notwo valves are open at the same time, to prevent cross-contamination ofthe sources. Such a condition can be imposed on the station bycomputerized means known in the art. In addition, computerized means canbe used to ensure that the reagents are dispensed in a desired sequence.

[0052] Although the invention is adaptable for use to distribute a smallamount of fluid in a variety of applications and to cover surfaces ofany size, the invention is particularly useful in lowering fluid wastein a device for forming biomolecular arrays. The geometry, sizeconstraints and other limitations of such a device make it difficult toemploy ordinary silkscreening methods that use a squeegee to coat asurface with a fluid. The ordinary biomolecular array device maycomprise a substrate having a contact area in any shape includingwithout limitation, square, rectangular, circular, etc. Substrateshaving a 3″ by 3″ inch contact area and substrates having a 6″ by 6″contact area have been produced. The distance between distributionmember and the contact area is ordinarily on the order of about 100microns. However, the distance may be as low as about 5 microns to ashigh as about 3000 microns and may be controlled by the height of thespacer, i.e., the distance between the generally parallel upper andlower surface of the spacer. The critical factor in determining thedistance is the volume of fluid or reagent needed to effect the desiredresult. That volume can be readily determined. As is apparent from FIG.1, the application volume is dependent on the contact area and thedistance between the distribution member and the contact area. To lowerthe amount of fluid waste, it is preferred that for any application of aparticular volume of the fluid, the volume does not substantially exceedthe application volume. It is particularly preferred that the volume ofthe fluid is no more than about 150% of the application volume.Optimally, the volume of the fluid is no more than about 110% of theapplication volume. In addition, the enclosure volume may correspond tothe application volume such that the enclosure volume does notsubstantially exceed the application volume.

[0053] The distribution member is now described. The distribution membermay be any of a variety of shapes. However, it is preferred that thedistribution member be generally planar with roughly parallel upper andlower surfaces. The distribution member must include a permeable portionwith a plurality of channels therethrough that provide communicationbetween the upper surface and the lower surface. Thus, the channelsterminate in openings on the upper and lower surfaces of thedistribution member. The cross-sectional area of the channels and theirterminal channels are typically small for a number of reasons. First, afluid dispensed on the upper surface will immediately pass through thepermeable portion of the distribution member if the channels are toolarge. User control of the rate and the manner of fluid distribution isthereby compromised. In addition, it is important to keep in mind theinvention may be adapted to apply a very small amount of fluid to coat asurface. Depending on the surface properties of a particular fluid andsubstrate surface, the fluid may tend to form beads on the substrate.Therefore, the lower surface of the distribution member may be used tomechanically ensure that the distributed fluid uniformly contacts thecontact area of the substrate surface. Holes with large diameters andexcessively long channels may render the distribution member incapableof serving this function. Possible versions of the distribution memberare illustrated in FIGS. 2A and 2B. FIG. 2A illustrates a flat piece 100that is perforated with circular channels 106 extending from oneparallel surface to another. FIG. 2B illustrates a distribution memberhaving a permeable portion comprising a mesh.

[0054] When a small amount of fluid is to be applied, surfaces forcesbecome increasingly important. Thus, the material of the distributionmember should be chosen according to the fluid or fluids to be used.While the invention may be adapted to apply any number of fluids,typical fluids that are used in the formation of biomolecular arraysinclude, but are not limited to, those that contain: water;acetonitrile; alcohols such as methanol, ethanol, propanol, isopropanol,and ethylene glycol; and ketones such as acetone and methyl ethylketone. In addition, such fluids may contain biomolecules such asoligonucleotides, polynucleotides, oligopeptides and polypeptides. As abasic requirement, the material from which the distribution member ismade must be dimensionally stable to exposure with the components of thefluid or fluids used, if the distribution member is to be used more thanonce. In addition, the material should obviously not impart anyundesired contaminants into fluid. For example, when acetonitrile isused as a solvent, the distribution member should not be made with aflat perforated piece of polystyrene, since polystyrene is soluble inacetonitrile. In addition, due to the quantity of fluid involved, thematerial should be selected to take advantage of its intrinsic surfaceproperties with respect to the fluid to be dispensed. For example, ifthe fluid contains water, the distribution member may be made from ahydrophobic material such as polytetrafluoroethylene to ensure thatfluid does not wick toward the distribution member from the contact areaof the substrate surface. As will be evident to one of ordinary skill inthe art, coatings may also be applied at specific areas on thedistribution member to selectively control the wetting properties of thespecific areas. As a general rule, ceramic and metallic materials aresuitable for use in distribution members for because they are stablewith respect to many fluids and it is possible to make constructperforated flat piece having channels with precision needed to renderthe invention operative. Certain polymers that are resistant to solventsmay also be a suitable material.

[0055] Variations of the foregoing will be apparent to thoseknowledgeable in the art. For example, if a plurality of fluids isdispensed, the apparatus may include more than one fluid transferchannel connected to the enclosure. In other words, variations known toone of ordinary skill in means of conveying fluids from their sources tothe enclosure may be employed. As another example, a sealing materialmay be provided and disposed between two components of the apparatus ifsealing contact is made. As still another example, fastening means maybe employed if two components of the invention are affixed to oneanother.

[0056] It is to be understood that while the invention has beendescribed in conjunction with the preferred specific embodimentsthereof, that the foregoing description is intended to illustrate andnot limit the scope of the invention. Other aspects, advantages andmodifications within the scope of the invention will be apparent tothose skilled in the art to which the invention pertains.

[0057] All patents, patent applications, and publications mentionedherein are hereby incorporated by reference in their entireties.

1. A method for applying a fluid onto a surface of a solid substrate,comprising the steps of: (a) providing a distribution member having anupper surface and a lower surface, and including a permeable portionhaving channels therethough; (b) positioning the distribution membersuch that the lower surface of the distribution member is in opposingrelation to the surface of the substrate; (c) dispensing the fluid ontothe upper surface of the permeable portion of the distribution membersuch that the fluid penetrates and is retained by the permeable portionof the distribution member; and (d) applying a distribution pressuredifferential between the upper and lower surfaces without using asqueegee such that a portion of the fluid passes through the channels ofthe permeable portion of the distribution member and onto the solidsubstrate surface.
 2. The method of claim 1, wherein step (c) comprisesapplying a loading pressure differential between the upper and lowersurfaces of the distribution member.
 3. The method of claim 1, wherein:step (a) further comprises providing a spacer having an upper surfaceand a lower surface; step (b) further comprises positioning the uppersurface of the spacer in contact with the lower surface of thedistribution member and about the permeable portion, and positioning thelower surface of the spacer on the solid substrate surface, therebydefining an application volume between the lower surface of thedistribution member and the substrate surface; and the fluid passes intothe application volume during step (d).
 4. The method of claim 1,wherein step (b) further comprises positioning the distribution memberin generally uniformly spaced relation to the substrate surface.
 5. Themethod of claim 1, wherein step (d) comprises raising pressure at theupper surface of the distribution member.
 6. The method of claim 1,wherein step (d) comprises lowering pressure at the lower surface of thedistribution member.
 7. The method of claim 1, further comprising thestep of: (e) flushing a second fluid through the permeable portion ofthe distribution member such that a portion of the fluid is displacedaway from the solid substrate surface.
 8. The method of claim 1, furthercomprising the step of: (e) flushing a gas through the permeable portionof the distribution member such that a portion of the fluid is displacedaway from the solid substrate surface.
 9. The method of claim 7, whereinthe gas comprises nitrogen or argon.
 10. The method of claim 1, whereinthe fluid comprises water, acetonitrile, an alcohol, an acid, a base, anoxidizing agent, a reducing agent, or a ketone.
 11. The method of claim1, wherein the fluid contains a biomolecule.
 12. The method of claim 11,wherein the biomolecule is an oligonucleotide, polynucleotide,oligopeptide, or polypeptide.
 13. The method of claim 3, wherein thefluid occupies a fluid volume that does not substantially exceed thanthe application volume.
 14. The method of claim 13, wherein the fluidvolume is no more than about 150% of the application volume.
 15. Themethod of claim 14, wherein the fluid volume of the fluid is no morethan about 110% of the application volume.
 16. The method of claim 1,wherein the distribution member is generally planar.
 17. The method ofclaim 1, wherein the distribution member is substantially flat andperforated.
 18. The method of claim 1, wherein the distribution membercomprises a mesh.
 19. An apparatus for applying a fluid onto a surfaceof a solid substrate, comprising: a distribution member having an uppersurface, a lower surface and a permeable portion formed by a pluralityof channels extending from the upper surface to the lower surface; anenclosure having an enclosure volume formed by an enclosing wall affixedabout the permeable portion in sealed contact with the upper surface ofthe distribution member; means for positioning the distribution memberin relation to the solid substrate surface such that the lower surfaceof the distribution member is in opposing relation to the solidsubstrate; means for introducing the fluid on to the upper surface ofthe distribution member within the enclosure; and squeegeeless means forproducing a pressure differential between the upper surface and thelower surface of the distribution member, whereby the pressuredifferential causes a portion of the fluid to pass through the memberand onto solid substrate surface.
 20. The apparatus of claim 19, furthercomprising a spacer having an upper surface and a lower surface, theupper spacer surface affixed to the lower surface of the distributionmember about the permeable portion and the lower spacer surface incontact with the substrate surface, such that an application spacehaving an application volume is substantially enclosed by the spacer,the lower surface of the distribution member and the substrate surface.21. The apparatus of claim 18 wherein the upper spacer surface and thelower spacer surfaces are generally parallel surfaces of the spacer 22.The apparatus of claim 20, further comprising an opening in the spacersuch that the application space fluidly communicates with open air. 23.The apparatus of claim 20, wherein the enclosure volume is notsubstantially greater than the application volume.
 24. The apparatus ofclaim 19, wherein the enclosure volume is no more than about 150% of theapplication volume.
 25. The apparatus of claim 24, wherein the enclosurevolume is no more than about 110% of the application volume.
 26. Theapparatus of claim 19, wherein the distribution member is generallyplanar.
 27. The apparatus of claim 19, wherein the distribution memberis substantially flat and perforated.
 28. The method of claim 19,wherein the distribution member comprises a mesh.
 29. The apparatus ofclaim 19, wherein the means for introducing the fluid comprises a fluidsource for supplying the fluid, a fluid transfer channel connected tothe enclosure and a fluid valve disposed between the fluid source andthe fluid transfer channel, where the fluid source is in fluidcommunication with the fluid transfer channel when the fluid valve isopen.
 30. The apparatus of claim 29, further comprising means forintroducing a second fluid, wherein the means for introducing the secondfluid comprises a second fluid source for supplying the second fluid anda second fluid valve disposed between the second fluid source and thefluid transfer channel, where the second fluid source is in fluidcommunication with the fluid transfer channel when the second fluidvalve is open.
 31. The apparatus of claim 29, wherein the squeegeelessmean for producing a pressure differential comprises a gas source forsupplying the gas and a gas valve disposed between the gas source andthe gas transfer channel, where the gas source is in fluid communicationwith the gas transfer channel when the gas fluid valve is open.
 32. Theapparatus of claim 19, wherein the squeegeeless means comprises meansfor raising pressure within the chamber.
 33. The apparatus of claim 32,wherein the pressure raising means comprises means for introducing a gasinto the enclosure from a pressured source.
 34. The apparatus of claim33, wherein the gas comprises nitrogen or argon.
 35. The apparatus ofclaim 19, wherein the fluid comprises water, acetonitrile, an alcohol,or a ketone.
 36. The apparatus of claim 19 wherein the fluid contains abiomolecule.
 37. The apparatus of claim 36, wherein the biomolecule isan oligonucleotide, polynucleotide, oligopeptide, or polypeptide.
 38. Anreagent application station, comprising: a distribution member having anupper surface, a lower surface and a permeable portion formed by aplurality of channels extending from the upper surface to the lowersurface; an enclosure formed by an enclosing wall affixed about thepermeable portion in sealed contact with the upper surface of thedistribution member; means for positioning the distribution member inrelation to the solid substrate surface such that the lower surface ofthe distribution member is in generally uniformly spaced oppositionrelation to the solid substrate; a source for each reagent; a variablepressure pump having an inlet for each source and an outlet in fluidcommunication with the enclosure; and a valve between each inlet andeach source.
 39. The reagent application station of claim 38, wherein notwo valves are open at the same time.
 40. A method for applying a fluidonto a surface of a solid substrate having a biomolecule attachedthereto, comprising the steps of: (a) providing a distribution memberhaving an upper surface and a lower surface, and including a permeableportion having channels therethough; (b) positioning the distributionmember such that the lower surface of the distribution member is inopposing relation to the surface of the substrate; (c) dispensing thefluid onto the upper surface of the permeable portion of thedistribution member such that the fluid penetrates and is retained bythe permeable portion of the distribution member; and (d) applying adistribution pressure differential between the upper and lower surfacessuch that a portion of the fluid passes through the channels of thepermeable portion of the distribution member and onto the solidsubstrate surface to contact with the biomolecules.
 41. The method ofclaim 40, wherein the biomolecule are covalently attached to thesubstrate surface.
 42. The method of claim 40, wherein the biomoleculerepresents a feature in an array attached to the substrate surface. 43.The method of claim 40, wherein the biomolecule is a nucleotidicbiomolecule or a peptidic biomolecule.